Understanding the mechanisms of cellular function and their dysfunction in disease requires a detailed picture of the molecular interactions in cells. In particular, it is desirable to have imaging tools with single-molecule sensitivity, molecular-scale resolution, and genome-scale capacity to allow direct visualization of these molecular interactions and to probe the collective behaviors of different genes and gene products that give rise to cell and tissue function. The long-term research goal of my laboratory is to develop advanced fluorescence imaging methods that meet these demands and to apply these methods to elucidate molecular mechanisms of cellular functions that are of both fundamental and medical significance. In the next five years, we propose to focus our NIGMS-funded research mainly in the following two directions. (1) To understand the structure and function of a novel cytoskeletal structure in neurons. Our NIGMS- supported research on the development and application of super-resolution STORM imaging has led to the discovery of a periodic membrane-associated cytoskeleton structure in neurons, primarily in axons. While we have a basic model of the ultrastructural organization of some key components of this structure, including actin, spectrin and associated molecules, we expect many additional protein components to be present in this structure. These components and their structural organization remain to be determined. Our understanding of the functional roles of this novel structure is also far from complete. We propose to use super-resolution imaging in conjunction with other methods to determine the protein components and structural organization of this periodic skeleton structure, and to investigate its functional roles in axon morphogenesis and synaptogenesis, action potential generation and propagation, axon degeneration, and other axonal functions. (2) To investigate the spatial organization of chromatin and chromosomes important for gene regulation. The spatial organization of chromatin plays an important role in many essential genome functions such as gene expression regulation, DNA replication, repair and recombination. In particular, many features of the chromatin conformation have been implicated in gene regulation, such as open and closed chromatin states, promoter- enhancer interactions, topologically-associated domains, and chromosome territories. Misregulation of chromatin structures has been implicated in a variety of diseases. However, many gaps remain in our understanding of the three-dimensional (3D) organization of chromatin and chromosomes. Our NIGMS- supported research on STORM imaging and chromatin remodeling studies allowed us to develop a super- resolution chromatin imaging approach and reveal novel chromatin organizations. We will advance our chromatin/chromosome imaging capability by developing the ability to trace the 3D path of the chromatin chain in the chromosome and to study questions on chromatin organization that are important for gene regulation.